LOCUS       sequence-309430-        8099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for chromosomal integrations of any sequence at a 
            locus downstream of the glmS gene in Enterobacteriaceae using 
            kanamycin resistance as a selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8099)
  AUTHORS   Holden ER, Wickham GJ, Webber MA, Thomson NM, Trampari E
  TITLE     Donor plasmids for phenotypically neutral chromosomal gene 
            insertions in Enterobacteriaceae.
  JOURNAL   Microbiology (Reading). 2020 Nov 23. doi: 10.1099/mic.0.000994.
  PUBMED    33226934
REFERENCE   2  (bases 1 to 8099)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Microbiology (Reading). 2020 Nov 23. doi: 10.1099/mic.0.000994."
COMMENT     Sequence Label: pDOC-K-glmS
FEATURES             Location/Qualifiers
     source          1..8099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1720..1739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1973..1990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2107..2128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2143..2148,2149..2166,2167..2173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2181..2197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2186..2208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2205..2221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2205..2221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(2724..2771)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2981..3775
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3035..3054)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3645..3664
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3966..3999)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(4523..4540)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4523..4539)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4532..4554)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4719..4736)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4889..4975
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5067..5094
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     oriT            5541..5650
                     /note="incP origin of transfer"
     promoter        6024..6469
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(6470..6556,6557..7891)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     primer_bind     complement(7930..7949)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8049..8071
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta gccaccactt caagaactct gtagcaccgc ctacatacct
     1441 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1501 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1561 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1621 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1681 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1741 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1801 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1861 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     1921 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     1981 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     2041 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     2101 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     2161 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     2221 ccatgattac gccaagctct agggataaca gggtaatcga tgaattcgct cgaaggcgcg
     2281 ctgaagctga aagagatctc ttacattcac gcggaagcct atgctgcagg cgagctgaaa
     2341 cacgggccgt tggcgctgat tgacgccgat atgccggtga ttgtggtggc gccgaacaac
     2401 gaactgctgg agaaactgaa atccaacatc gaagaagttc gcgcacgtgg cggtcagttg
     2461 tacgtcttcg ccgatcagga tgcgggtttc gtcagcaacg acaacatgca catcatcgag
     2521 atgccgcatg tggaagaggt cattgcgcct atcttctaca ccgttccgct gcaactgctg
     2581 gcgtatcacg tggcgctgat taaaggcacc gacgttgacc agccgcgtaa cctggcgaaa
     2641 tcggttaccg tagagtaagt acaaatgtag taccaggcag aaggccgggt acctaggacc
     2701 ggtcaattgg ctggagctgc ttcgaagttc ctatactttc tagagaatag gaacttcgga
     2761 ataggaactt caagatcccc cacgctgccg caagcactca gggcgcaagg gctgctaaag
     2821 gaagcggaac acgtagaact taagggaatt gccagctggg gcgccctctg gtaaggttgg
     2881 gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg
     2941 atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3001 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3061 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3121 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3181 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3241 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3301 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3361 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3421 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3481 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     3541 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     3601 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     3661 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgctct acggtatcgc
     3721 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     3781 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     3841 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3901 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccag cttcaaaagc
     3961 gctctgaagt tcctatactt tctagagaat aggaacttcg gaataggaac taaggaggat
     4021 attcatatct cgagcatatg cccggggcgg ccgcaagctt tcgacagacg gccttttttt
     4081 gtgcgctgtg acaggcgctg ttctttctct atggtggctg aagagcgact aagaggtgct
     4141 acgcctgcct gcgctacggg cacccggtca cattctcttc tttaaggccc tggctcaatc
     4201 caggtggatc ggaaatcaaa ccaccccagg ttattcaggt ggatccccct ggcccggtcg
     4261 gggcttttga gtcgcatcca gtggtgaaaa agcggctgta gccagcctga acgggtggtt
     4321 tccctgacca gttgttccgc gctgatggtt tcggcatgcc actgggtttc ccattgggcc
     4381 agcagcgcat catccccacc actgatggcg tggcgcagta aaggggagcc cagtagccat
     4441 gtaccgacgt tccactcttc gggaatgggg aaattgaccg gctagcgtcg actagtaggg
     4501 ataacagggt aatgagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4561 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4621 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     4681 gagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     4741 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     4801 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     4861 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     4921 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     4981 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     5041 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt
     5101 tctacaaact cttttgttta tttttctaaa tacattcaaa tatgcatgcg cctgatgcgg
     5161 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatcgacatc cgccctcacc
     5221 gccaggaacg caaccgcagc ctcatcacgc cggcgcttct tggccgcgcg ggattcaacc
     5281 cactcggcca gctcgtcggt gtagctcttt ggcatcgtct ctcgcctgtc ccctcagttc
     5341 agtaatttcc tgcatttgcc tgtttccagt cggtagatat tccacaaaac agcagggaag
     5401 cagcgctttt ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat
     5461 ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg
     5521 tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt
     5581 tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga
     5641 ggctggccgg ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag
     5701 ccaaccagga agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg
     5761 attgaggaaa aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc
     5821 cagggctaca aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc
     5881 aatggcgacc tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc
     5941 acggcgcggt tcggtgatgc cacgatcctc gccctgctgg cgaagatcga ctctagctag
     6001 aggatcgatc ctttttaacc catcacatat acctgccgtt cactattatt tagtgaaatg
     6061 agatattatg atattttctg aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga
     6121 aactataaaa aatacagaga atgaaaagaa acagatagat tttttagttc tttaggcccg
     6181 tagtctgcaa atccttttat gattttctat caaacaaaag aggaaaatag accagttgca
     6241 atccaaacga gagtctaata gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga
     6301 taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt atactttggc gtcacccctt
     6361 acatatttta ggtctttttt tattgtgcgt aactaacttg ccatcttcaa acaggagggc
     6421 tggaagaagc agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa
     6481 aaagtttgca aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc
     6541 aactcaagcg tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc
     6601 ccatattaca cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca
     6661 agttcctgag ttcgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt
     6721 ttgggacagc tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca
     6781 catcgtcttt gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt
     6841 ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa
     6901 agacagcgac aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc
     6961 aggttcagcc acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg
     7021 taaacattac ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag
     7081 ctctttgaac atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac
     7141 gtatcaaaat gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac
     7201 gctgagagat cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa
     7261 cactggaact gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg
     7321 caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg
     7381 cacggctgag ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact
     7441 gaaaaaagtg atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc
     7501 gaacgtcttt aaaatgaacg gcaaatggta tctgttcact gactcccgcg gatcaaaaat
     7561 gacgattgac ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt
     7621 aactggccca tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc
     7681 taacgatgta acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt
     7741 cgtgattaca agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc
     7801 gcctagcttc ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct
     7861 tgaacaagga caattaacag ttaacaaata aaaacgcaaa agaaaatgcc gattatggtg
     7921 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     7981 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     8041 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcga
//
